The present invention relates to novel analogues of Monocyte Chemoattractant Protein-1 (MCP-1) and corresponding potynucleotide sequences vectors. host cells and recombinant expressions particularly in E. coli. 
MCP-1 is a member of the chemokine family of pro-inflammatory cytokines which mediate leukocyte chemotaxis and activation. MCP-1 is a Cxe2x80x94C chemokine which is one of the most potent and selective T-cell and monocyte chemoattractant and activating agents known. MCP-1 has been implicated in the pathophysiology of a large number of inflammatory diseases including rheumatoid arthritis (RA), glomerular nephritides, lung fibrosis, restenosis (International Patent Application WO 94/09128), alveolitis (Jones et al. 1992, J. Immunol. 49, 2147) and asthma. Other disease areas where MCP-1 is thought to play a part in their pathology are: atherosclerosis (e.g. Koch et al. 1992 J. Clin. Invest. 90, 772-779); psoriasis (Deleuran et al. 1996 J. Dermatological Science 13, 228-236), delayed -type hypersensitivity reactions of the skin; inflammatory bowel disease (Grimm et al. 1996 J. Leukocyte Biol. 59, 804-812), multiple sclerosis and; brain trauma (Berman et al. 1996 J. Immunol. 156, 3017-3023). An MCP-1 inhibitor may also be useful to treat stroke, reperfusion injury, ischemia, myocardial infarction and transplant rejection.
It is known that analogues of MCP-1 can be prepared which are antagonistic to its biological properties. Rollins in U.S. Pat. No. 5,459,128 describes MCP-1 antagonists which include truncation at the N-terminus of the protein. Gong in J. Exp. Med. (1995) 181, 631, describes a particular MCP-1 antagonist of this type which is MCP1(9-76) (SEQ ID NO: 30). Gong made MCP-1(9-76) (SEQ ID NO: 30) by direct chemical synthesis. Chemical synthesis is not a commercially attractive process for manufacture of MCP-1(9-76) (SEQ ID NO: 30). Lewis and Gong in Canadian patent application 2152141 describe chemically synthesised MCP-1 analogues which are truncated at the N-tenninus. Conservative amino acid substitutions for some amino acids (excluding valine) are also described therein (see page 7) but preferably only at regions beyond position 35 of MCP-1.
A recombinant method would be preferable for large scale commercial manufacture of MCP1(9-76) (SEQ ID NO: 30) but to date there has been no specific experimental disclosure of any such recombinant methodology.
A preferred organism for recombinant manufacture is E. coli because of its ease of handling. However, Rollins U.S. Pat. No. 5,459,128 warns of problems with use of bacterial expression systems due to incorrect protein folding and states a preference for COS cell expression systems (see column 2, lines 49-51). However, COS cells are not desirable for large scale manufacture because they only provide a transient expression system. We have discovered in our experiments (which are presently unpublished) that secretory expression (i.e. using a leader sequence to direct expressed protein through the cytoplasmic membrane) of MCP1(9-76) (SEQ ID NO: 30) in E. coli has the disadvantage of giving poor yields (see Comparative Example 2). Furthermore we have also discovered that when a polynucleotide encoding MCP1(9-76) is expressed intracellularly in E. coli the resulting product is undesirably heterogeneous due to the presence of an additional methionine residue at the N-terminus in a significant proportion (typically 50%) of the product. There thus exists a need for an improved way of making a MCP1(9-76) (SEQ ID NO: 30) type protein, particularly in E. coli. 
The present invention is based on the discovery that substitution of an alanine, glycine or threonine for the natural valine in position 9 of MCP1(9-76) gives a novel MCP-1 analogue (termed xe2x80x9c[V9A]MCP1(9-76)xe2x80x9d (SEQ ID NO: 9), xe2x80x9c[V9G]MCP1(9-76)xe2x80x9d (SEQ ID NO: 26) or xe2x80x9c[V9T]MCP1(9-76)xe2x80x9d (SEQ ID NO: 29) respectively herein) which can be recombinantly manufactured in E. coli to produce a substantially homogeneous product (at least substantially lacking unwanted methionine at the N-terminus) in good yield which retains the antagonistic effect of MCP1(9-76) as measured in the chemotaxis assay described herein.
According to one aspect of the present invention there is provided a protein selected from [V9A]MCP1(9-76) (SEQ ID NO: 9), [V9G]MCP1(9-76) (SEQ ID NO: 26) or [V9T]MCP1(9-76) (SEQ ID NO: 29). The protein sequences of [V9A]MCP1(9-76), [V9G]MCP1(9-76) and [V9T]MCP1(9-76) are set out in SEQ ID NO: 9, 26 and 29 respectively. Preferably the protein is selected from [V9A]MCP1(9-76) (SEQ ID NO: 9) or [V9G]MCP1(9-76) (SEQ ID NO: 26) and of these [V9A]MCP1(9-76) (SEQ ID NO: 9) is especially preferred.
Note Lewis and Gong in Canadian patent application 2152141 describe conservative amino acid substitutions in truncated MCP-1 analogues but these are preferably beyond position 35 (of MCP-1) and furthermore that disclosure is completely silent on substitution of valine anywhere in the molecule. Preferably the [V9A]MCP1(9-76) (SEQ ID NO: 9), [V9G]MCP1(9-76) (SEQ ID NO: 26) or [V9T]MCP1(9-76) (SEQ ID NO: 29) is essentially free of methionine at its N-terminus. The term xe2x80x9cessentially freexe2x80x9d in this context means that methionine is present at a level of about 10% or less (when) analysed by any one of reverse phase HPLC, capillary zone electrophoresis, Edman degradation and electrospray mass spectrometry) at the N-terminus of [V9A]MCP1(9-76) (SEQ ID NO: 9), [V9G]MCP1(9-76) (SEQ ID NO: 26) or [V9T]MCP1(9-76) (SEQ ID NO: 29), preferably methionine is present at a level of less than 5% at the N-terminus of [V9A]MCP1(9-76) (SEQ ID NO: 9), [V9G]MCP1(9-76) or [V9T]MCP1(9-76) (SEQ ID NO: 29), preferably methionine is present at a level of less than 3% at the N-terminus of [V9A]MCP1(9-76) (SEQ ID NO: 9), [V9G]MCP1(9-76) (SEQ ID NO: 26) or [V9-76) (SEQ ID NO: 29), more preferably methionine is present at a level of less than 1% at the N-terminus of [V9A]MCP1(9-76) (SEQ ID NO: 9), [V9G]MCP1(9-76) (SEQ ID NO: 26) or [V9T]MCP1(9-76) (SEQ ID NO: 29), more preferably methionine is present at a level of less than 0.3% at the N-terminus of [V9A]MCP1(9-76) (SEQ ID NO: 9), [9G]MCP1(9-76) (SEQ ID NO: 26) or [V9T]MCP1(9-76) (SEQ ID NO: 29) and more preferably methionine is present at a level of less than 0.1% at the N-terminus of [V9A]MCP1(9-76) (SEQ ID NO: 9), [V9G]MCP1(9-76) (SEQ ID NO: 26) or [V9T]MCP1(9-76) (SEQ ID NO: 29). Preferably the [V9A]MCP1(9-76) (SEQ ID NO: 9), [V9G]MCP1(9-76) (SEQ ID NO: 26). [V9T]MCP1 (9-76) (SEQ ID NO: 29) gives a single peak when analysed by reverse phase HPLC.
According to another aspect of the invention there is provided a polynucleotide sequence encoding a protein selected from [V9A]MCP1(9-76) (SEQ ID NO: 9), [V9G]MCP1(9-76) (SEQ ID NO: 26) OR [V9T]MCP1(9-76) (SEQ ID NO: 29). The polynucleotides of the present invention may be in the form of RNA or in the form of DNA including synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptide may be identical to any coding sequence shown herein or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same, mature polypeptides. The polynucleotides which encode the mature polypeptide may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a leader sequence; and optionally non-coding sequence 5xe2x80x2 and/or 3xe2x80x2 of the coding sequence for the mature polypeptide. Thus, the term xe2x80x9cpolynucleotide sequence encoding a protein selected from [V9A]MCP1(9-76) (SEQ ID NO: 9), [V9G]MCP1(9-76) (SEQ ID NO: 26) or 76) (SEQ ID NO: 29)xe2x80x9d encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence. Examples of DNA sequences which encode [V9A]MCP1(9-76) (SEQ ID NO: 9), [V9G]MCP1(9-76) (SEQ ID NO: 26) and [V9T]MCP1(9-76) (SEQ ID NO: 29) are set out in SEQ ID NO: 8, 25 and 28 respectively.
In this specification amino acid analogues of specific amino acid sequences are contemplated which retain the relevant biological properties of a component of the invention but differ in sequence by one or more conservative amino acid substitutions, deletions or additions. However the specifically listed amino acid sequences are preferred and analogues should preferably be modified at corresponding positions after position 35 of MCP-1. Truncation at the C-terminus is also contemplated, preferably at corresponding positions after position 52 of MCP-1. Typical conservative amino acid substitutions are tabulated below.
In this specification nucleic acid variations (deletions, substitutions and additions) of specific nucleic acid sequences are contemplated which retain the ability to hybridise under stringent conditions to the specific sequence in question. Stringent conditions are defined as 6xc3x97SSC, 0.1% SDS at 60xc2x0 C. for 5 minutes. However specifically listed nucleic acid sequences are preferred. It is contemplated that peptide nucleic acid may be an acceptable equivalent of polynucleotide sequences, at least for purposes that do not require translation into protein (Wittung (1994) Nature 368, 561).
According to another aspect of the invention there is provided a vector comprising a polynucleotide sequence encoding a protein selected from [V9A]MCP1(9-76) (SEQ ID NO: 9), [V9G]MCP1(9-76) (SEQ ID NO: 26) or [V9T]MCP1(9-76) (SEQ ID NO: 29). Preferably the vector is an expression vector, more preferably the vector is for use in E. coli, especially designed for intracellular expression in E. coli. Intracellular expression in E. coli can be in soluble form and/or insoluble form. If expressed in insoluble form, for example as inclusion bodies, the protein can be solubilised/refolded by conventional techniques. Expression as inclusion bodies is especially preferred. An especially preferred vector is pZT7#3.3 comprising a polynucleotide sequence encoding aprotein selected from [V9A]MCP1(9-76) (SEQ ID NO: 9), [V9G]MCP1(9-76) (SEQ ID NO: 26) or [V9T]MCP1(9-76) (SEQ ID NO: 29).
Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation, initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli. As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis., USA). These pBR322 xe2x80x9cbackbonexe2x80x9d sections are combined with an appropriate promoter and the structural sequence to be expressed. Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well known to those skilled in the art.
According to another aspect of the invention there is provided a host cell comprising a vector as described herein. Preferably the host is E. coli. 
According to another aspect of the invention there is provided a method of making a protein selected from [V9A]MCP1(9-76) (SEQ ID NO: 9), [V9G]MCP1(9-76) (SEQ ID NO: 26) or [V9T]MCP1(9-76) (SEQ ID NO: 29) which comprises culture of a host as described herein in a culture medium under conditions for expression of the protein and optionally at least partially purifying the protein.
According to another aspect of the present invention there is provided a pharmaceutical composition comprising a protein of the invention described herein in association with a pharmaceutically-acceptable diluent or carrier, optionally in a form suitable for intravenous administration.
According to another aspect of the present invention there is provided a protein as described herein for use as a medicament.
According to another aspect of the present invention there is provided use of a protein as described herein for preparation of a medicament for treatment of inflammatory diseases.
It will be appreciated that the dose and dosage regimen will depend upon the particular effector moiety employed, the population of the target cell and the patient""s history. The dose of the protein administered will typically be in the range 0.1 to 10 mg/kg of patient weight.
The proteins of the present invention will normally be administered in the form of a pharmaceutical composition. Thus according to the present invention there is also provided a pharmaceutical compostion which comprises a protein (as defined herein) in association with a pharmaceutically-acceptable diluent or carrier. An example of such a formulation is given herein in Example 5.
Pharmaceutical compositions of the present invention may be formulated in a variety of dosage forms. Generally, the proteins of the present invention will be administered parenterally, preferably intravenously. A particular parenteral pharmaceutical composition is one which is formulated in a unit dosage form which is suitable for administration by injection. A parenteral composition is preferably a solution in isotonic saline or isotonic dextrose, buffered if necessary to a pH of 5 to 9.
Alternatively, the parenteral composition may be one designed for slow release in which case the amount of protein per unit dose is in general greater than that required when a conventional injectable formulation is used. A preferred slow release formulation is a continuous release formulation, for example a fomulation of the type described in European Patent Specification No. 58481. A preferred slow release parenteral formulation contains from 20 to 150 mg of polypeptide per unit dose. Systemic delivery of peptides and proteins is described in Banga et al. (1988), Int. J. Therapeutics 48: 15-50.
Topical administration is also contemplated e.g. for treatment of psoriasis and also administration by inhalation e.g. for treatment of IPF or asthma.
Particularly suitable compositions comprise a solution, emulsion or suspension of the protein in association with a pharmaceutically acceptable parenteral carrier or diluent. Suitable carriers or diluents include aqueous vehicles, for example water or saline, and non-aqueous vehicles, for example fixed oils or liposoines. The compositions may include agents which enhance the stability of the protein in the composition. For example, the composition may include a buffer. The concentration of the protein will vary, but in general, the composition of the invention will normally be administered such that a daily parenteral dose, will be about 10-300 mg for a 70 kg human.
For further information on Formulation the reader is referred to Chapter 25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990. For further information on Routes of Administration and Dosage Regimes the reader is referred to Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board). Pergarnon Press 1990.
According to a further aspect of the present invention there is provided a protein of the invention for use as a medicament.
According to a further aspect of the present invention there is provided use of a protein of the invention in preparation of a medicament for antagonising an MCP-1 mediated effect in a mammal, especially man. Particular MCP-1 mediated effects include inflammatory diseases, especially rheumatoid arthritis, multiple sclerosis and atherosclerosis.
According to a further aspect of the present invention there is provided a method for antagonising an MCP-1 mediated effect in a mammal, such as man, in need of such treatment, which comprises administering to said mammal an effective amount of a pharmaceutical composition of the invention.
Amino acid nomenclature is set out below.
Abbreviations used herein include: